In vitro regeneration and Agrobacterium mediated transformation of Turkish commercial barley (Hordeum vulgare L.)

Nahid Hazrati, Ali Ergul, Farzad Nofouzi, Saime Unver, Khalid Mahmood Khawar, Mohammad Hasanzadeh

Abstract: The aim of the work was to create Agrobacterium-based transformation method for barley and use this technology to produce stable transgenic plants so; the sterilized seeds of the barley Ozen genotype were primed in 4 different nutrient media. Regeneration test was then carried out using embryo explants of germinated seeds in medium containing seven hormone levels of NAA (naphthalene acetic acid) + BAP (benzyl amino purine). According to the results, priming factor was significant (P<0.01) for all measured traits except for root number per explant and hormone factor was significant for all measured traits except for leaf area trait. According to the mean comparisons, priming medium containing 20 mg/l BAP was selected as the best one. The nutrient medium containing 0.1 mg/l NAA+0.45 mg/l BAP showed the highest plant growth factors. To investigate genetic transformation of barley Ozen genotype, zygotic embryos (control embryos and embryos treated with cold treatment for 24 hours on MS medium) were treated with Agrobacterium tumefaciens GV 2260 strain harboring p35 GUS-INT plasmid followed by co-cultivation for 24 and 48 hours and then explants transferred to the selection medium. Survived plants were planted in peat soil and the leaves of developing and acclimated plants were subjected to GUS histochemical analysis and polymerase chain reaction (PCR). The highest yields were obtained in the explants subjected to 24 hours cold treatment followed by co-cultivation for 48 hours so that, the percent of green plants (17.18%), percent of plants with positive GUS (15.6%) and percent of plants with positive PCR (14.06%), had the highest values.

Keywords: Agrobacterium tumefaciens; Barley; GUS; PCR; transformation

Date published: 2019-12-19